JPH05244956A - Production of l-phenylalanine by recombinant escherichia coli - Google Patents

Abstract

PURPOSE: To enable the production of L-phenylalanine in high yield.

CONSTITUTION: A new Escherichia coli (KCCM 10,013) capable of producing L-phenylalanine in a high yield is used. The method for producing L- phenylalanine by recombinant Escherichia coli which may be transformed with a novel plasmid pMW16 containing two promoters and a temperature-sensitive repressor for expressing a pheA gene and an aroF gene wherein chorimate mutase p-prephenate dehydratase is coded for by the pheA gene and 3-deoxy-D- arabinoheptulosonate 7 phosphate synthase is coded for by the aroF gene.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to recombinant E. coli (recombina
nt Escherichia coli) (hereinafter, simply referred to as Escherichia coli) . More specifically, the present invention relates to a novel Escherichia coli containing a plasmid for producing L-phenylalanine and a method for producing L-phenylalanine using the novel microorganism.

[0002]

2. Description of the Related Art L-Phenylalanine is one of the essential amino acids and can be used for the synthetic production of a sweetener, aspartame (ASPARTAME: registered trademark). L using microorganisms
-Many synthetic methods of phenylalanine are known. For example, JP-A 37-6345 and JP-A 60-1608.
90 is a Brevibacterium or Corynebac
terium sp. ) for producing L-phenylalanine. JP 55-165797 discloses a similar method using E. coli that requires tyrosine and is resistant to tryptophan analogues. Korean Patent Laid-Open 88-11331 and Korean Laid-Open 88-11
332 is a tyrosine and tryptophan auxotrophy (a
uxotroph) is a revertant,
And a similar method using E. coli resistant to phenylalanine, tyrosine and tryptophan analogues is disclosed. Korean Patents 89-3682 and 89-3714 disclose rate-limiting enzymes.
Disclosed is a method for producing L-phenylalanine by recombinant Escherichia coli in which the copy number of the gene encoding the gene is amplified.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a novel E. coli (KCCM 10,01) capable of producing L-phenylalanine in high yield.
The purpose is to provide 3).

Another object of the present invention is the pheA gene and a
Novel plasmid pMW1 containing two promoters for expressing the roF gene and a temperature sensitive repressor
It is to provide a method for producing L-phenylalanine using recombinant Escherichia coli which can be transformed with 6. Chorismate mutase p-prephenate dehydratase is present in the pheA gene, and 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (
3-deoxy-D-arabinoheptulosonate ・ 7-phosphate syntha
se) are each encoded by the aroF gene.

A further object of the present invention is the high yield of L-
It is to provide a replicable recombinant plasmid pMW16 capable of producing transformed Escherichia coli capable of producing phenylalanine.

A further object of the present invention is to provide a method for producing L-phenylalanine in high yield, which comprises culturing the Escherichia coli of the present invention in a medium.

Other objects and further scope of application of the present invention will become apparent from the detailed description of the present invention given below. However, changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from the following detailed description, and thus the detailed description and the specific examples showing the preferred embodiments of the present invention are merely illustrative. Should be understood as shown as.

The invention is better understood by the detailed description given below and the drawings which are given merely by way of illustration, but it goes without saying that the invention is not limited thereto.

The aroF gene and the pheA gene for use in the production of L-phenylalanine are derived from Escherichia coli MW.
It is derived from PWJ 304 (KCCM 10,013).

FIG. Referring in detail to the drawings prepared for explaining the present invention such as 1., the method for producing L-phenylalanine using the recombinant plasmid is controlled by the enzymatic action in E. coli cells. One of the enzymes controlling the reaction is chorismate mutase p-prephenate dehydratase. One of the other enzymes that control the reaction is 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (hereinafter, "DAH
PHP synthase "). DAHP synthase is three isoenzymes encoded by aroF gene, aroG gene, and aroH gene, respectively.
It exists as (isoenzyme). In the present invention, phe
The P _L promoter of λ phage is linked to each gene in order to extremely increase the expression of A gene and aroF gene, and a temperature sensitive repressor is used to control the expression of the gene.

FIG. Plasmid pMW as shown in 2
The preparation method of 16 is the Recombinant DNA Methodology and
It is described in Molecular Cloning. Plasmid pM
The recombinant method for preparing W16 is described in Molecular Clonin
g, A Laboratory Manual (T. Mariatis et al.) and C
urrent Protocols in Molecular Biology (Frederick
M. Ausubel et al.).

In this way a new strain MWPWJ 304 for use in the production of L-phenylalanine is obtained. According to the provisions of the Budapest Treaty, the new strain MWPWJ 304 was purchased on December 20, 1991 at the Korean Culture Center of Microorganisms.
rean Culture Collection of Microorganisms) and was given accession number KCCM 10,013.

The present invention is described in further detail in connection with the following examples, which are merely specific examples.
It goes without saying that the present invention is not limited to this.

[0014]

Example 1 Preparation of plasmid pMW16 (1) Isolation of pMW12 DNA pMW12 (US Pat. Nos. 5,008,190 and 5,030,567), which are recombinant plasmids containing the pheA gene and the aroF gene. Medium) for 16 hours at 37 ° C. (medium salt restriction enzyme buffer).
enzyme buffer) (50mM sodium chloride, 10m
Cleavage with EcoRV in M Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM dithiothreitol). Cleaved DNA
Was treated with a phenol-chloroform mixture and precipitated with ethanol to yield linearized plasmid DNA (linearized
plasmid DNA) was obtained. The plasmid DNA was used for calf intestinal alkaline phosphatase.
phosphatase) (hereinafter abbreviated as “CIP”),
Linear plasmid DNA self-ligating
Prevented.

(2) Preparation of P _L promoter fragment Plasmid pPL _C 2833 was cleaved with restriction enzymes HaeII and BamHI, and a 0.26 Kb P _L fragment was recovered from a low melting point agarose gel and purified. The P _L fragment of 0.26Kb having cohesive ends, 20 minutes reaction buffer at 11 ° C. (Tris -HCl of 50 mM (pH 8.
0) 5 mM magnesium chloride, 5 mM dithiothreitol, 50 μg / ml bovine serum albumin (BS
A), 100 μM dATP, 100 μM dGTP,
Blunt ends were formed by treatment with T ₄ DNA polymerase in 100 μM dCTP and 100 μM dTTP).

(3) Preparation of plasmid pMW14 The linear plasmid pMW12 treated with phosphatase in the above step (1) was mixed with the P _L promoter fragment obtained in the above step (2) having blunt ends at a ratio of 1: 3. Mix at a ratio of T ₄ D for 16 hours at 16 ° C
The fragments were ligated by treatment with NA ligase. The ligated recombinant plasmid was transformed into Escherichia coli MW which requires L-phenylalanine for growth according to a conventional calcium chloride method.
It was transformed into EC203-7. The transformed strain was treated with MM medium containing 50 μg / ml of the antibiotic kanamycin (glucose 10 g, ammonium sulfate 4 g,
Monobasic potassium phosphat
e) 2 g, thiamine-HCl 1 mg, fumaric acid 0.5
g, agar 20 g, distilled water 1, pH 7.4). The recombinant plasmid pMW14 was added to kanamycin 50
Separated from cultures grown in MM medium containing μg / ml.

(4) Preparation of pMW15 Plasmid pM containing temperature sensitive repressor CI ₈₅₇
After treating K5 with the restriction enzyme BglII, a 0.9 Kb DNA fragment was recovered from a 0.9% agarose gel and subjected to T ₄
Treatment with DNA polymerase forms blunt ends. The plasmid pMW14 obtained in the above step (3) in which the P _L promoter was ligated to the front end of the aroF gene was Dra.
Cleavage with III and treatment with CIP to remove the 5'-phosphate group. The treated plasmid pMW14
Mixed with CI ₈₅₇ fragment having blunt ends at both ends to give T ₄ D
It was ligated with NA ligase. The recombinant plasmid was incorporated into Escherichia coli HB101 to obtain pMW15.

(5) Preparation of pMW16 The isolated pMW15 recombinant plasmid was cleaved with BamHI and BglII and treated with T ₄ DNA ligase to obtain pMW16. The pMW15 plasmid contains all tyrA
Contains genes. In pMW16, the 0.7 Kb fragment of the tyrA gene was removed. The pMW16
The recombinant plasmid was transferred to a temperature-sensitive tyrosine leaky strain (tyrosine
-leaky strain), that is, Escherichia coli MWWJ 304
(KFCC10737, deposited at Corean Federation of Culture Collection on August 30, 1991), and kanamycin at 50 μg / ml
It was cultured at 37 ° C. for 24 hours in the containing medium. pMW16
Among the strains into which M. aureus was incorporated, the strain producing the best L-phenylalanine, MWPWJ 304 (KCCM10,
013, deposited on December 20, 1991, with the Corean Culture Collection of Microorganisms. The new strain MWPWJ 304 (KCC
The biochemical properties of M 10,013) are
Same as J 304 (KFCC 10,737).
However, compared to the host strain MWWJ 304, the novel strain MWPWJ 304 requires more tyrosine for growth and produces more L-phenylalanine. Then, the production amount of L-phenylalanine and DAHP synthase activity of the new strain MWPWJ 304 increased.

Experimental Example 1 Compared with the existing strain, the enzyme activity and L-phenylalanine production of the new strain MWPWJ 304 are increased as shown below (Tables 1 and 2): Table 1 DAHP synthase enzyme activity Strain Culture temperature Enzyme activity unit MWPEC 13-60 32 ° C 1.0 (KCTC 8337P) MWWJ 304 37 ° C 0.9 (KFCC 10,737) MWPWJ 304 37 ° C 3.1 (KCCM 10,013) The above data is eye. ． The method of I. Shiio et al (Journal of Biochemistry
Biochemistry), 75, 987-997, 1974). MWPWJ 304 (KCCM 1
The enzyme activity of 0,013) is MWPEC 13-60.
(KCTC 8337P) based on enzymatic activity.
MWWJ 304 (KFCC 10,737) and MW
The PWJ 304 (KCCM 10,013) medium contained 10 when compared to the MWPEC 13-60 medium.
An extra amount of 0 mg / l L-tyrosine was included.

Example 2 Preparation of L-Phenylalanine (A) Strain MWPWJ 304 (KCCM 10,013).

(B) Medium glucose 3% tryptone 1% bactoyeast extract 1% sodium chloride 0.1% kanamycin 10 mg / l pH 7.0.

(C) Fermentation medium glucose 6% potassium sulfate 0.04% ammonium sulfate 2% sodium citrate 0.05% fumaric acid 0.05% magnesium chloride 0.08% monobasic potassium phosphate 0.1% dibasic Potassium phosphate 0.1% Bactose yeast extract 0.1% Monosodium glutamate 0.05% Cobalt chloride 0.1 mg / l Zinc sulfate 1 mg / l Manganese chloride 2 mg / l Calcium chloride 5 mg / l Ferric chloride 20 mg / l L-tyrosine 300 mg / l thiamine hydrochloride 10 mg / l nicotinic acid 10 mg / l pH 7.0.

(D) Fermentation method 40 ml of medium is put into a 500 ml test flask, and 1
It was autoclaved at 20 ° C. for 20 minutes. After sterilization,
New E. coli strain MWPWJ 304 (KCCM10,01
3) was inoculated into a flask and shake-cultured at 37 ° C. for 16 hours. The fermentation medium was prepared according to the above-mentioned recipe. Thereafter, separately autoclaved 3.5% calcium carbonate was added to the fermentation medium, and further 2 ml of seed broth was added, and the medium was vigorously stirred and fermented at 37 ° C. for 36 hours. .. After the fermentation was completed, the amount of accumulated L-phenylalanine was 16.2 g / l.

Example 3 Preparation of L-Phenylalanine (C) The strain (A), (B) medium, and (C) fermentation medium were the same as in Example 1 except that L-tyrosine in the fermentation medium was 400 mg / l. The same as that used in 2 and 2 was used.

(D) Fermentation method 1 l of fermentation medium was placed in a 2 l fermentor and the mixture was stirred at 120 ° C. for 1 hour.
It was autoclaved for 5 minutes. New stock MWPWJ 3
04 (KCCM 10,013) was added to 50 ml of medium in a 500 ml flask, and shake culture was performed at 37 ° C. for 16 hours. 50 ml of culture broth
roth) to the fermentor, 1,000 rpm and 1.0
Fermentation was carried out at 37 ° C. for 48 hours under vvm (oxygen ratio) conditions. During the fermentation, a pH value of 7.0 was maintained by adding ammonium hydroxide, and 60% glucose solution was added to the fermentor three times when the glucose concentration was below 1%.

The total amount of glucose used during fermentation was 18
It was 5 g / l. L-phenylalanine is 50.8g
/ L concentration was obtained. 1 l of fermentation solution was purified by a conventional method such as adsorption on an ion exchange resin and isolation using ammonium hydroxide, and 45.7 g /
l of L-phenylalanine was obtained.

Table 2 Yield of L-phenylalanine (g / l) Strain culture temperature MWWJ 304 MWPWJ 304 (° C) (KFCC 10,737) (KCCM 10,013) 31 14.9 13.7 34 20.6 21.2 37 31.5 50.8 39 15.5 22.8 * 200 mg / l L-tyrosine was added to the fermentation medium of MWWJ 304 (KFCC 10,737).

Example 4 The procedure of Example 3 was repeated except that 27 l of the culture broth was placed in a 50 l fermentor and 1.3 l of culture broth was placed in the fermenter under the conditions of 450 rpm and 1.0 vvm.
The amount of L-phenylalanine obtained was 49.1 g / l.

Although the present invention has been described above,
It goes without saying that various modifications are possible. These modifications are not considered to depart from the spirit and scope of the invention, and all such modifications that are obvious to those skilled in the art are intended to be covered by the following claims.

[Brief description of drawings]

FIG. 1 is a FIG. FIG. 1 is a diagram showing a biosynthetic metabolic pathway of aromatic amino acids in Escherichia coli. In the figure, feedback inhibition means inhibition of enzyme activity, and feedback inhibition suppresses synthesis of an enzyme that acts at the transcription level from DNA to mRNA.

FIG. 2 FIG. FIG. 2 is a diagram showing a preparation process of the recombinant plasmid pMW16 and its restriction map.

[Explanation of symbols]

1 DAHP synthase (suppressing tyrosine) encoded by aroF gene 2 DAHP synthase (suppressing phenylalanine) encoded by aroF gene 3 DAHP synthase (suppressing tryptophan) encoded by aroF gene 4 Chorismate mutase encoded by pheA gene p- Prephenate dehydratase 5 tyrA gene-encoded chorismate mutase p-prephenate dehydratase 6 trpE gene-encoded anthranilate
Synthase

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location // (C12N 1/21 C12R 1:19) (C12P 13/06 C12R 1:19) (72) Inventor Don June Lee South Korea Seoul Theodaemun-Kunam Gadjiwa Dong 353-20 (72) Inventor Chang Hee Won South Korea Seoul Sung Buk Jung-Gleung-2-Don 559-29 (72) Inventor Byun Lak Lim South Korea Seoul Nowon -Ku Jong Gye Dong Gun Yang 2-Cha Apert 106-1507 (72) Inventor Honkyu Choi South Korea Seoul Donda Emun Kuk Changlian Guri Dong Dong Hyundai Part 3-405

Claims (8)

[Claims] 1. A pheA gene encoding chorismate mutase p-prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate.
Ar encoding 7-phosphate synthase
A replicable recombinant plasmid comprising plasmid DNA containing two promoters for expressing the oF gene and a temperature-sensitive repressor, which is transformed into E. coli and has the optimal L-phenylalanine-producing ability. It is possible to obtain Escherichia coli MWPW
A recombinant plasmid identified as plasmid pMW16 contained in J 304 (KCCM 10,013). 2. The replicable recombinant plasmid according to claim 1, wherein the promoter is a λP _L promoter. 3. The replicable recombinant plasmid according to claim 1, wherein the temperature sensitive repressor is the CI ₈₅₇ repressor. 4. Escherichia coli MWPWJ 304 (KCCM 10,01) having an optimal L-phenylalanine-producing ability because it has a pheA gene and an aroF gene.
3). 5. A method of culturing in a medium an E. coli strain carrying the plasmid from KCCM 10,013.
A method for producing L-phenylalanine. 6. The method for producing L-phenylalanine according to claim 5, wherein the culture is performed in the presence of sugar. 7. The method for producing L-phenylalanine according to claim 6, wherein the sugar is glucose. 8. The method for producing L-phenylalanine according to claim 6, wherein the culture is performed at 37 ° C.